This invention relates in general to microorganisms and more particularly to a process and apparatus for enumerating microorganisms in a water sample.
Contamination of public waters such as streams, rivers and lakes by residential and industrial sewage constitutes a major environmental problem, since such waters are the primary source of drinking water and the water used for the preparation and processing of foods, drugs and cosmetics. Moreover, such bodies of water represent a principal source of recreation. While the solution to the contamination problem of course involves eliminating harmful microorganisms from effluents which enter such bodies of water; it also requires continuous microbiological monitoring of the effluents as well as the public waters themselves. Current techniques for microbiological analysis are time-consuming, laborious and expensive.
There are two widely practiced procedures for ascertaining the bacterial count of water or aqueous suspensions. The first, commonly called the Most Probable Number, or MPN procedure, requires serial 10-fold dilutions of a sample into a nutrient medium with each successive dilution usually being 1 ml. of the previous dilution mixed into 9 ml. of nutrient medium. The microorganisms are then allowed to grow and the various dilutions are observed for turbidity, change of color, or some other indication of growth. The reciprocal of the next to the last dilution showing growth is taken as the concentration in cells per milliter.
In the second procedure cells are collected on a membrane filter, and the filter is placed either on an absorbent pad saturated with culture media, or on solid media containing bacteriological agar. The cells are visualized as colonies on the membrane filter after a defined period of incubation and the number of colonies, related to the dilution used and amount filtered, provides the number of cells in the original sample. This procedure is commonly called the membrane filter process.
Since the membrane filter procedure is limited to samples which will not clog the membrane pores, and is not effective when chlorinated samples are tested, the MPN procedure has become the most widely accepted procedure for determining bacterial counts in water.
In its simplest form, the MPN procedure involves a straight 10-fold dilution to extinction using a single set of test tubes. Measured aliquots of these dilutions are then added to tubes containing nutrient medium. For example, the first dilution tube will contain 10 ml. of sample without any dilution (10.degree. dilution). The second dilution tube will contain 1 ml. of sample and 9 ml. of diluent (10.sup.-.sup.1 dilution). The third dilution tube will contain 0.1 ml. of sample in 9.9 ml. of diluent (10.sup.-.sup.2 dilution). The fourth, fifth, etc. dilution tubes are filled with 9 ml. diluent and 1 ml. of suspension derived from their immediate predecessors in the series. Thus, the concentration of sample in the fourth dilution tube will be 1/1000 that of the original sample (10.sup.-.sup.3 dilution), while the concentration in the fifth dilution tube will be 1/10000 that of the sample (10.sup.-.sup.4 dilution).
One ml. aliquots of each dilution tube are then added to corresponding tubes containing nutrient medium (culture tubes), thus resulting in a series of tubes. The first culture tube of this series will contain 1.0 ml. of the 10.degree. dilution, the second tube 1.0 ml. of the 10.sup.-.sup.1 dilution, and so on to a final tube containing 1.0 ml. of the 10.sup.-.sup.5 dilution. These culture tubes are then incubated and observed for changes. If the fifth culture tube (which received 1.0 ml. of the 10.sup.-.sup.5 dilution) is the last to exhibit growth, then at least one cell was present in the 1 ml. derived from the 10.sup.-.sup.5 dilution of the dilution tube and added to the fifth culture tube. It is assumed, therefore, that each ml. in the sixth dilution tube contained one cell also, or a concentration therein of one cell/ml. Since this dilution constitutes 1/100,000 of the 10.degree., or 10.sup.-.sup.5, then the concentration in the 10.degree. sample is the reciprocal of 10.sup.-.sup.5 which is 10.sup.5 cells per ml.
A single series of tubes gives only a rough estimate. To provide greater accuracy, usually 5 tubes of each dilution are employed. Under normal circumstances not all of the series will dilute to extinction at the same dilution. For example,three of the tubes of 10.sup.-.sup.4 dilution may show growth, while the two remaining do not. Tables prepared from statistical evaluations are available to provide the bacteria count for any combination of growth and no growth tubes at various dilutions. Of course, the greater the number of series, the more accurate is the count.
From the foregoing, it is quite apparent that the MPN method with all its dilutions of dilutions, is quite laborious and time-consuming and requires a considerable amount of culture medium.